The present invention relates to methods of identifying candidate compounds for regulating skeletal muscle mass or function or regulating the activity or expression of a corticotropin releasing factor-2 receptor (CRF2R). The invention also relates to methods for the treatment of skeletal muscle atrophy or methods for inducing skeletal muscle hypertrophy using CRF2R as the target for intervention and to methods of treating muscular dystrophies using CRF2R and corticotropin releasing factor-1 receptor (CRF1R) as targets.
There are two corticotropin releasing factor receptors, identified to date (CRF1R and CRF2R) which belong to G-protein coupled receptor (GPCR) class. Agonist activation of CRF1R or CRF2R leads to Gxcex1s activation of adenylate cyclase. Adenylate cyclase catalyzes the formation of cAMP, which in turn has multiple effects including the activation of protein kinase A, intracellular calcium release and activation of mitogen-activated protein kinase (MAP kinase). In other studies, the enhancement of intracellular inositol triphosphate synthesis, after agonist activation of CRF receptors, suggests that CRFRs also couple to Gxcex1q.
CRF1R and CRF2R have been cloned from human, rat, mouse, chicken, cow, catfish, frog and sheep. CRF1R and CRF2R each have a unique distribution patterns. In humans three isoforms, alpha, beta and gamma, of the CRF2R receptor have been cloned. Homologs for alpha and beta CRF2R have been identified in rat.
Several ligands/agonists of the CRFRs are known. Corticotropin releasing factor (or hormone, CRF or CRH) binds to and activates CRF1R and CRF2R. CRF is a major modulator of the body""s responses to stress. This 41-amino acid peptide presides over a panoply of neuronal, endocrine, and immune processes as the primary regulator of the hypothalamus-pituitary-adrenal hormonal axis (HPA axis). In addition, there is substantial sequence homology between CRF and the amphibian peptide sauvagine as well as the telostian peptide urotensin, both of which act as agonists of CRF1R and CRF2R. These three peptides have similar biological properties as hypotensive agents and ACTH secretogogues. In addition, a mammalian congener of urotensin, urocortin, has been characterized.
The CRF receptors can be distinguished, from non-CRFRs, pharmacologically through the use of receptor selective agonists and antagonists. These selective agonists and antagonist, along with the CRFR knockout mice, have been useful in determining which CRF receptor mediates specific biological responses.
The role of CRF1R has been fairly well established. Mice in which the CRF1R gene has been ablated (CRF1R knockout) demonstrate an impaired stress response and reduced anxiety-like behavior. CRF1R is a major mediator of the HPA axis. Specifically, corticotropin releasing factor, which is released from the hypothalamus and transported to the anterior pituitary via the hypothalamic-hypophysial portal system, interacts with the CRF1R present on cells located in the anterior pituitary. Agonist activation of the CRF1R results in release of ACTH from the cells of the anterior pituitary into the systemic circulation. The released ACTH binds the ACTH receptor present on cells located in the adrenal cortex, resulting in the release of adrenal hormones including corticosteroids. Corticosteroids mediate many effects including, but not limited to, immune system suppression via a mechanism which involves thymic and splenic atrophy. Thus activation of the CRF1R indirectly results in the down-regulation of the immune system via activation of the HPA axis.
The role of CRF2R is less well developed. Mice in which the CRF2R gene has been ablated (CRF2R knockout) demonstrate an impaired food intake reduction following stimulation with urocortin, lack of vasodilation, but a normal stress response. Experiments with CRF2R demonstrated that CRF2R is responsible for the hypotensive/vasodilatory effects of CRFR agonists and for the reduction in food intake observed following treatment of mice with CRFR agonists.
Skeletal muscle is a plastic tissue which readily adapts to changes in either physiological demand for work or metabolic need. Hypertrophy refers to an increase in skeletal muscle mass while skeletal muscle atrophy refers to a decrease in skeletal muscle mass. Acute skeletal muscle atrophy is traceable to a variety of causes including, but not limited to: disuse due to surgery, bed rest, or broken bones; denervation/nerve damage due to spinal cord injury, autoimmune disease, or infectious disease; glucocorticoid use for unrelated conditions; sepsis due to infection or other causes; nutrient limitation due to illness or starvation; and space travel. Skeletal muscle atrophy occurs through normal biological processes, however, in certain medical situations this normal biological process results in a debilitating level of muscle atrophy. For example, acute skeletal muscle atrophy presents a significant limitation in the rehabilitation of patients from immobilizations, including, but not limited to, those accompanying an orthopedic procedure. In such cases, the rehabilitation period required to reverse the skeletal muscle atrophy is often far longer than the period of time required to repair the original injury. Such acute disuse atrophy is a particular problem in the elderly, who may already suffer from substantial age-related deficits in muscle function and mass, because such atrophy can lead to permanent disability and premature mortality.
Skeletal muscle atrophy can also result from chronic conditions such as cancer cachexia, chronic inflammation, AIDS cachexia, chronic obstructive pulmonary disease (COPD), congestive heart failure, genetic disorders, e.g., muscular dystrophies, neurodegenerative diseases and sarcopenia (age associated muscle loss). In these chronic conditions, skeletal muscle atrophy can lead to premature loss of mobility, thereby adding to the disease-related morbidity.
Little is known regarding the molecular processes which control atrophy or hypertrophy of skeletal muscle. While the initiating trigger of the skeletal muscle atrophy is different for the various atrophy initiating events, several common biochemical changes occur in the affected skeletal muscle fiber, including a decrease in protein synthesis and an increase in protein degradation and changes in both contractile and metabolic enzyme protein isozymes characteristic of a slow (highly oxidative metabolism/slow contractile protein isoforms) to fast (highly glycolytic metabolism/fast contractile protein isoforms) fiber switch. Additional changes in skeletal muscle which occur include the loss of vasculature and remodeling of the extracellular matrix. Both fast and slow twitch muscle demonstrate atrophy under the appropriate conditions, with the relative muscle loss depending on the specific atrophy stimuli or condition. Importantly, all these changes are coordinately regulated and are switched on or off depending on changes in physiological and metabolic need.
The processes by which atrophy and hypertrophy occur are conserved across mammalian species. Multiple studies have demonstrated that the same basic molecular, cellular, and physiological processes occur during atrophy in both rodents and humans. Thus, rodent models of skeletal muscle atrophy have been successfully utilized to understand and predict human atrophy responses. For example, atrophy induced by a variety of means in both rodents and humans results in similar changes in muscle anatomy, cross-sectional area, function, fiber type switching, contractile protein expression, and histology. In addition, several agents have been demonstrated to regulate skeletal muscle atrophy in both rodents and in humans. These agents include anabolic steroids, growth hormone, insulin-like growth factor I, and beta adrenergic agonists. Together, these data demonstrate that skeletal muscle atrophy results from common mechanisms in both rodents and humans.
While some agents have been shown to regulate skeletal muscle atrophy and are approved for use in humans for this indication, these agents have undesirable side effects such as hypertrophy of cardiac muscle, neoplasia, hirsutism, androgenization of females, increased morbidity and mortality, liver damage, hypoglycemia, musculoskeletal pain, increased tissue turgor, tachycardia, and edema. Currently, there are no highly effective and selective treatments for either acute or chronic skeletal muscle atrophy. Thus, there is a need to identify other therapeutic agents which regulate skeletal muscle atrophy.
Muscular dystrophies encompass a group of inherited, progressive muscle disorders, distinguished clinically by the selective distribution of skeletal muscle weakness. The two most common forms of muscle dystrophy are Duchenne and Becker dystrophies, each resulting from the inheritance of a mutation in the dystrophin gene, which is located at the Xp21 locus. Other dystrophies include, but are not limited to, limb-girdle muscular dystrophy which results from mutation of multiple genetic loci including the p94 calpain, adhalin, xcex3-sarcoglycan, and xcex2-sarcoglycan loci; fascioscapulohumeral (Landouzy-Dejerine) muscular dystrophy, myotonic dystrophy, and Emery-Dreifuss muscular dystrophy. The symptoms of Duchenne muscular dystrophy, which occurs almost exclusively in males, include a waddling gait, toe walking, lordosis, frequent falls and difficulty in standing up and climbing stairs. Symptoms start at about 3-7 years of age with most patients confined to a wheelchair by 10-12 years and many die at about 20 years of age due to respiratory complications. Current treatment for Duchenne muscular dystrophy includes administration of prednisone (a corticosteroid drug), which while not curative, slows the decline of muscle strength and delays disability. Corticosteroids, such as prednisone, are believed to act by blocking the immune cell activation and infiltration which are precipitated by muscle fiber damage resulting from the disease. Unfortunately, corticosteroid treatment also results in skeletal muscle atrophy which negates some of the potential benefit of blocking the immune response in these patients. Thus, there is a need to identify therapeutic agents which slow the muscle fiber damage and delay the onset of disability in patients with muscular dystrophies, but cause a lesser degree of skeletal muscle atrophy than current therapies.
One problem associated with identification of compounds for use in the treatment of skeletal muscle atrophy or of muscular dystrophies has been the lack of good screening methods for the identification of such compounds. Applicants have now found that CRF2Rs are involved in the regulation of skeletal muscle mass or function and that agonists of CRF2Rs are able to block skeletal muscle atrophy and/or induce hypertrophy of skeletal muscle. The present invention solves the problem of identifying compounds for the treatment of muscle atrophy by providing screening methods using CRF2R which can be used to identify candidate compounds useful for the treatment of muscle atrophy. The present invention also solves the problem of finding compounds for treatment of muscle dystrophies by providing a screening method to identify candidate compounds which activate both the CRF1R and CRF2R.
The present invention relates to the use of CRFRs to identify candidate compounds that are potentially useful in the treatment of skeletal muscle atrophy and or to induce skeletal muscle hypertrophy. In particular, the invention provides in vitro methods for identifying candidate compounds for regulating skeletal muscle mass or function comprising contacting a test compound with a cell expressing CRF2R, or contacting a test compound with isolated CRF2R, and determining whether the test compound either binds to or activates the CRF2R. Another embodiment of the invention relates to a method for identifying candidate therapeutic compounds from a group of one or more candidate compounds which have been determined to bind to or activate CRF2R comprising administering the candidate compound to a non-human animal and determining whether the candidate compound regulates skeletal muscle mass or muscle function in the treated animal.
A further embodiment of the invention relates to a method for identifying candidate compounds for regulating skeletal muscle mass or function comprising, in any order: (i) contacting a test compound with a cell expressing a functional CRF2R, and determining a level of activation of CRF2R resulting from the test compound; (ii) contacting a test compound with a cell expressing a functional CRF1R, and determining the level of activation of CRF1R resulting from the test compound; followed by (iiii) comparing the level of CRF2R activation and the level of CRF1R activation; and (iv) identifying those test compounds that show similar activity toward CRF2R and CRF1R or show selectivity for CRF2R as candidate compounds for regulating skeletal muscle mass or function.
The invention further provides methods for identifying candidate compounds that prolong or augment the agonist-induced activation of CRF2R or of a CRF2R signal transduction pathway. These methods comprise in any order or concurrently: (i) contacting a test compound; with a cell which expresses functional CRF2R (ii) treating the cell with a CRF2R agonist for a sufficient time and at a sufficient concentration to cause desensitization of the CRF2R in control cells; followed by (iii) determining the level of activation of CRF2R and identifying test compounds that prolong or augment the activation of a CRFR or a CRFR signal transduction pathway as candidate compounds for regulating skeletal muscle mass or function. In a particular embodiment, the present invention relates to a method of identifying candidate therapeutic compounds from a group of one or more candidate compounds determined to prolong or augment the activation of a CRF2R or of a CRF2R signal transduction pathway comprising: administering the candidate compound, in conjunction with a CRF2R agonist, to a non-human animal and determining whether the candidate compound regulates skeletal muscle mass or function in the treated animal.
The invention further provides methods for identifying candidate compounds that increase CRF2R expression comprising contacting a test compound with a cell or cell lysate containing a reporter gene operatively associated with a CRF2R gene regulatory element and detecting expression of the reporter gene. Test compounds that increase expression of the reporter gene are identified as candidate compounds for increasing CRF2R expression. In a particular embodiment, the present invention relates to a method of determining whether those candidate compounds which increase CRF2R expression can be used to regulate skeletal muscle mass or function in vivo by administering a candidate compound to a non-human animal and determining whether the candidate compound regulates skeletal muscle mass or function in the treated animal.
The invention further provides methods for identifying candidate compounds that increase CRF expression comprising contacting a test compound with a cell or cell lysate containing a reporter gene operatively associated with a CRF gene regulatory element and detecting expression of the reporter gene. Test compounds that increase expression of the reporter gene are identified as candidate compounds for increasing CRF expression. In a particular embodiment, the present invention relates to a method of determining whether those candidate compounds which increase CRF expression can be used to regulate skeletal muscle mass or function in vivo by administering a candidate compound to a non-human animal and determining whether the candidate compound regulates skeletal muscle mass or function in the treated animal.
The present invention also relates to the use of CRF2R agonists, expression vectors encoding a functional CRF2R, expression vectors encoding a constitutively active CRF2R or compounds that increase expression of CRF2R, or CRF to treat skeletal muscle atrophy. In particular, the invention provides methods of treating skeletal muscle atrophy, in a subject in need of such treatment, comprising administering to the subject a safe and effective amount of a CRF2R agonist, an expression vector encoding a functional CRF2R, an expression vector encoding a constitutively active CRF2R, an expression vector encoding a CRF or CRF analog, or a compound that increases expression of CRF2R, or CRF. In a particular embodiment, the present invention relates to a method for treating skeletal muscle atrophy in a subject in need of such treatment comprising administering to the subject a safe and effective amount of a CRF2R agonist in conjunction with a safe and effective amount of a compound that prolongs or augments the agonist-induced activation of CRF2R, or of a CRF2R signal transduction pathway.
The present invention also relates to the use of a CRF2R agonist to increase skeletal muscle mass or function in a subject. In particular, the invention provides methods of increasing skeletal muscle mass or function in a subject in which such an increase is desirable, comprising identifying a subject in which an increase in muscle mass or function is desirable and administering to the subject a safe and effective amount of a CRFR agonist.
The invention further provides for pharmaceutical compositions comprising a safe and effective amount of a CRF2R agonist and a pharmaceutically-acceptable carrier. In a particular embodiment the pharmaceutical composition comprises a chimeric or human antibody specific for a CRF2R. In another particular embodiment the pharmaceutical composition comprises a CRF or CRF analog, preferably urocortin II.
The present invention also provides for antibodies to CRF2R and in particular to chimeric or human antibodies that are agonists of CRF2R.
Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Each of the CRFR nucleotide and protein sequences or CRF analog protein sequence included in the sequence listing, along with the corresponding Genbank or Derwent accession number(s) and animal species from which it is cloned, is shown in Table I. Also shown are accession numbers for related nucleotide sequences that encode identical, or nearly identical, amino acid sequences as the sequence shown in the sequence listing. These related sequences differ mainly in the amount of 5xe2x80x2 or 3xe2x80x2 untranslated sequence shown.